I am at a stage in my research where I have to extract the residual substrates in cultures I am using for biodegradation experiments. Previously, I start the extraction by adding 2ml bezene immediately I stop the degradation in the cultures at the different degradation time points ( I stop the degradation at the different time points by adding about 500uL of 6M sulphuric acid). Thereafter, I keep the cultures in a shaker(50 rpm) in a cooling room of about 10 degrees for about two days. At the end of the second day, I remove the benzene extracts assumed to contain the residual substrates(phenols) in the cultures and I measure the substrates concentration using the GC-MS. Now, I expect a high peak for time points of 0, 40, 50 and 60 percent degradation time points but what I see is that there are more or less no visible peaks. I am new to using the GC-MS and extraction and would like to get suggestions on how increase my extraction efficiency and to resolve this issue of getting invisible peaks.
Dear Precious,
what is the GC column that are you using and the composition of the sample that you are injecting in the GC-MS (pure benzene or diluted with solvent)? And are you sure to not have acid in your injection? This will damage the column
If is 100% dimethylpolysiloxane phase, the column is right for both benzene and phenols. You need to work on oven program (ramp and temperatures), column flow and split ratio. If you let me know these parameters and the amount that you are injecting in the column, we can find a solution. Furthermore, if you are doing a splitless mode, I think you should check if you can inject entirely benzene without problems, or if is better mix a half or more with methanol
Hi Andrea, the GC column I am using is J & W ZB1 60m X320um X 1um. The samples injected are benzene extracts that should contain phenols that have been extracted by the culture bottle. There are no acids in the sample.
Hi Precious,
consider derivatising your phenols, the derivates usually chromatograph considerably better. TMS is a possibility TMSH another one.
However in my world small peaks as such is very relative, I guess you need to calibrate you GC-MS with standard solutions and compare your extracts to that.
From Your question I am insecure whether you have low response (signal) in the GC-MS, or whether you have low recovery rates (extraction efficiency). If the extraction efficiency is low you should consider making a better contact of the solvent with the biomass. (more vigorous shaking, getting the biomass off and extract with heat (Soxhlet).
However the last possibility is: your bugs eat the phenols... phenols are pretty good carbon sources and easily mineralised or metabolised/incorporated in the biomass.
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